Influenza virus particles are covered by two types of surface glycoproteins, one is phytohemagglutinin (i.e., H), and the other is neuraminidase (i.e., N). Type H is divided into 15 subtypes, and type N is divided into 9 subtypes.
All human avian influenza viruses can cause avian influenza, but not all of the avian influenza viruses can cause human influenza. In the avian influenza viruses, H5, H7 and H9 can infect human, in which H5 is highly pathogenic, and H5N1 is a subtype of avian influenza virus. H5N1 is newly added to the list of infectious diseases, the occurrence of which, as stipulated by the regulations in the Infectious Disease Prevention Act from the Ministry of Public Health, must be reported, also known as highly pathogenic human avian influenza. Basing on their pathogenicity, avian influenza can be divided into highly pathogenic and low pathogenic avian influenza, and H5N1 belongs to the highly pathogenic avian influenza viruses.
In 1997, the H5N1 virus was isolated and identified for the first time from an influenza death case sample of a 3-year-old child in Hong Kong, China. By 2012, most of the human cases of infection have been directly from birds, only individual cases have been considered being from infected persons, and it is generally believed that the H5N1 virus has not been able to establish a sustained human-to-human transmission chain. Patients with severe avian influenza are generally infected with the subtype H5N1 virus. The patients show acute onset, and their early-stage performance is similar to that of common influenza, mainly including fever, with the body temperature being constantly above 39° C. and the course being 1-7 days, usually 3-4 days, accompanied by symptoms like runny nose, nasal congestion, cough, sore throat, headache, muscle aches and general discomfort. Some patients may have nausea, abdominal pain, diarrhea, watery stools and other digestive tract symptoms. Critical patients experience rapid development of the disease, which may lead to pneumonia, acute respiratory distress syndrome, pulmonary haemorrhage, pleural effusion, pancytopenia, renal failure, sepsis, shock and other complications. The total number of leukocytes is generally not increased, or even lowered. The critical patients generally have decreased total number of leukocytes and lymphocytes, accompanied with thrombocytopenia.
H5N1 subtype avian influenza virus is a type of virus that has the strongest infectivity and the highest lethality and is most prevalent in the avian influenza viruses found so far, which has been epidemic in many countries of the world.
Presently there is no effective and specific therapies for this disease, and vaccination is an effective preventive measure and key link. Therefore, the development of a safe, efficient vaccine with low-cost becomes one of the current hotspots of avian influenza prevention and control, and the vaccine development has made a lot of research achievements. However, since the virus mutates fast and has a variety of variants, the research and development speed cannot guarantee the effective resistance against viral variants; therefore, further researches on H5N1 invasion process and development of new therapies are more pressing tasks.
Micro ribonucleic acids (microRNA, or miRNA for short) are a class of non-coding, single stranded, small ribonucleic acid molecules with 19-23 nucleotides in length. They are highly conserved in evolution, and widely present in cells. Micro ribonucleic acids inhibit the translation of target mRNAs by recognizing an non-translated sequence at the 3′ end of the target mRNA and complementing not completely thereto. Owing to the diversity of the sequence, structure, abundance and expression, the micro ribonucleic acid becomes a powerful regulator for messenger RNAs and plays an unimaginably important role in the field of gene expression regulation.
Micro ribonucleic acids are closely related to many normal physiological activities of animals, involving in development, tissue differentiation, apoptosis, energy metabolism and other aspects of life activities of biological individuals. Moreover, micro ribonucleic acids are also inextricably linked with the occurrence and development of many diseases, and when a certain disease occurs, the expression of some micro ribonucleic acids are always up-regulated, and some are down-regulated.
Relevant studies have shown that, after infecting host cells, viruses or microorganisms will encode specific miRNAs that act on the host immune-related mRNAs to regulate the host cytokine system of the host and further affect the immune regulation.
In summary, in order to prevent and treat avian influenza more effectively, there is an urgent need to develop relevant reagents and methods for the detection and treatment of avian influenza.
Contents of the Invention
The object of the present invention is to provide relevant reagents and methods for effective detecting and treating of avian influenza.
In the first aspect of the present invention, provided is an isolated miRNA selected from:
(i) miRNA with a sequence as shown in SEQ ID NO: 1, 2 or 3, or
(ii) miRNA with a length of 20-26 nt and a core sequence as shown in SEQ ID NO.: 4; or
(iii) miRNA complementary to the said nucleotide sequence of miRNA in (i) or (ii).
In another preferred example, said “complementary” includes “substantially complementary” (the number of non-complementary bases ≤3, preferably ≤3, more preferably ≤1) and “fully complementary”.
In another preferred example, said miRNA is from avian influenza virus.
In another preferred example, said miRNA is from avian influenza virus H5N1.
In another preferred example, said miRNA is isolated from blood, body fluids or tissue samples of human or non-human mammals.
In another preferred example, said blood is plasma and/or serum.
In another preferred example, said non-human mammals are mice, rats, rabbits, pigs, bovine, sheep, etc.
In another preferred example, said miRNA is isolated from human.
In the first aspect of the present invention, provided is an isolated or artificially constructed precursor miRNA which can be cut and expressed as the miRNA of the first aspect of the present invention in animal cells.
In another preferred example, said animal cells include human cells.
In the third aspect of the present invention, provided is an isolated polynucleotide which can be transcribed in an animal cell into a precursor miRNA which can be cut and expressed into the miRNA as said in the first aspect of the present invention.
In another preferred example, said polynucleotide has the structure of formula I:Seqforward-X-Seqreverse   Formula I
In the formula I,
Seqforward is a nucleotide sequence that can be expressed as said miRNA in an animal cell;
Seqreverse is a nucleotide sequence substantially complementary or fully complementary to Seqforward;
X is a spacer sequence between Seqforward and Seqreverse, and said spacer sequence is not complementary to Seqforward and Seqreverse;
and after being transferred into the human cell, the structure shown in the formula I forms a secondary structure shown in formula II:

In the formula II, Seqforward, Seqreverse and X are defined above, and ∥ indicates the complementary base pairing relationship between Seqforward and Seqreverse.
In the fourth aspect of the present invention, provided is a vector comprising the miRNA of the first aspect, or the polynucleotide of the second aspect of the present invention.
In the fifth aspect of the present invention, provided is use of the miRNA of the first aspect of the present invention for: (a) the preparation of a reagent, a detecting chip or a kit for the detection of avian influenza; (b) the preparation of a regulator for regulating the PCBP2 expression or activity; and (c) the preparation of reagents for regulating the expression of cytokines.
In another preferred example, said regulator for regulating PCBP2 expression or activity is an inhibitor for down-regulating the PCBP2 expression or activity.
In another preferred example, said cytokines include TNFα, IFN-β, IL-6, IL-1β, or a combination thereof.
In the sixth aspect of the present invention, provided is a nucleic acid chip (e.g., a miRNA chip), comprising:
a solid-phase carrier; and
oligonucleotide probes orderly fixed on said solid-phase carrier, said oligonucleotide probes specifically capturing the miRNA of the first aspect of the present invention.
In the seventh aspect of the present invention, provided is use of the nucleic acid chip of the sixth aspect of the present invention for the preparation of a kit for the detection of avian influenza.
In the eighth aspect of the present invention, provided is a kit comprising the nucleic acid chip of the seventh aspect or the miRNA of the first aspect of the present invention.
In the ninth aspect of the present invention, provided is an inhibitor specifically inhibiting or blocking the miRNA of the first aspect of the present invention.
In another preferred example, said inhibitor is a miRNA sponge, or an antisense nucleic acid or a small molecule compound complementary to the miRNA sequence.
In another preferred example, said inhibitor is a nucleic acid (e.g., RNA, DNA or the like) complementary to the nucleotide sequence of the miRNA of (i) or (ii).
In the tenth aspect of the present invention, provided is use of the inhibitor for said miRNA of the ninth aspect of the present invention for the preparation of (a) a medicament for treating avian influenza, (b) a medicament for relieving the symptoms of avian influenza, (c) a medicament for reducing the quantity of avian influenza virus in a host animal, (d) a medicament for reducing the death rate of avian influenza, and (e) a medicament for reducing overactive immune responses.
In the eleventh aspect of the present invention, provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an inhibitor for specifically inhibiting or blocking the miRNA of the first aspect of the present invention.
In another preferred example, said miRNA inhibitor comprises a miRNA sponge, and an antisense nucleic acid complementary to the sequence of miRNA.
In the twelfth aspect of the present invention, provided is a method for screening medicament candidates for treating avian influenza, comprising the steps of:
(a) providing a test group and a control group, wherein to the said test group, a candidate substance is applied to cells or animals of the test group, and the expression level of miR-HA-3p in said test group is detected after the application, and in said control group, the same conditions as the test group are applied, without applying the candidate substance to cells or animals of the control group; and
(b) comparing the expression level of miR-HA-3p in the test group with that in the control group;
wherein if the expression level of miR-HA-3p in the test group is significantly lower than that in the control group, it is indicated that this candidate substance is a medicament candidate for treating avian influenza.
In another preferred example, the sequence of said miR-HA-3p is as shown in SEQ ID NOs: 1-3.
In another preferred example, said animals include mice, and said cells include cells cultured in vitro.
In the thirteenth aspect of the present invention, provided is a use of miR-HA-3p for the preparation of a regulator or a pharmaceutical composition for down-regulating the PCBP2 expression or activity.
In another preferred example, the sequence of said miR-HA-3p is as shown in SEQ ID NOs: 1-3.
It should be understood that all of the various technical features described above and specifically described hereinafter (such as examples) can be combined with one another within the scope of the present invention, so as to form new or preferred technical solutions. Due to space limitations, these are no longer tired out one by one.
In the figures, “Mut” represents the mutant, “control” represents the control, “Marker” represents the molecular weight standard, and “Mock” represents the blank control.